Rna polymerase mutants with increased thermostability

ABSTRACT

The present application relates to mutated RNA polymerases from bacteriophages that have increased stability, for example under high temperature conditions. Preferred mutated RNA polymerases according to the invention are mutant RNA polymerases from T7 or SP3 bacteriophages. An especially preferred embodiment of the present invention is a T7 RNA polymerase with a serine to proline amino acid change in the protein at position 633 of the amino acid sequence.

[0001] The present application relates to mutated RNA polymerases frombacteriophages that have increased stability, for example under hightemperature conditions. One example of bacteriophage encoded RNApolymerase is the T7 RNA polymerase. T7 is a bacteriophage capable ofinfecting E. coli cells. Examples of other E. coli infecting T7-likebacteriophages are T3, φI, φII, W31, H, Y, A1, croC21, C22 and C23. Anexample of a Salmonella typhimurium infecting bacteriophage is SP6.

[0002] The RNA polymerases of bacteriophages have high selectivity fortheir own promoter sequence. The T7 RNA polymerase will bind the T7 RNApolymerase promoter sequence but not one of the other bacteriophagepromoter sequences. The high promoter specificity ensures that thebacteriophage transcription reaction is only directed to its own genomeand not the host genome. The entire nucleotide sequence of the T7bacteriophage is known and the phage RNA polymerase is encoded by T7gene 1. Other RNA polymerases that resemble the T7 RNA polymerase arethe RNA polymerases of bacteriophages SP6 and T3. The T3 RNAP showsabout 80% homology with the T7 RNAP.

[0003] The T7 gene 1 has been cloned and expressed in bacteria allowingthe production of large quantities of the enzyme (Studier et al., U.S.Pat. No. 4,952,496). The T7 polymerase is a single chain protein of 883amino acids with a molecular weight of 98,6 Kda. T7 RNA polymerase doesnot require any auxiliary factors for accurate transcription. The enzymealone is capable of recognizing it's promoters, initiatingtranscription, elongating the RNA transcript and terminatingtranscription. T7 RNA polymerase is very efficient in transcribing DNAfrom its own promoters and elongates RNA five times faster compared toE. coli RNA polymerase. Their selectivity, activity and ability toproduce complete transcripts make the polymerases from bacteriophagesvery useful for a variety of purposes.

[0004] The present invention is concerned with the RNA polymerases ofT7-like bacteriophages that have been mutated.

[0005] Some specific mutants of T7-like bacteriophage RNA polymeraseshave been described. For example, in WO91/05866 an alternativeexpression system is described. The system is an attempt to use thebacteriophage T7 promoters to direct the transcription of a cloned genein bacteria. The system uses a truncated T7 RNA polymerase, the gene ofwhich is mutated by deleting a nucleotide (one or more basescorresponding to base 3809 and 3877 of a wild type T7 polymerase gene).This deletion results in a frame shift and consequently a newtranslation stop codon is created. In U.S. Pat. No. 5,385,834, a mutantT7 RNAP is also described. The mutant described in U.S. Pat. No.5,385,834 is a G to A transition at nucleotide 664 of T7 gene 1 thatconverts glutamic acid (222) to lysine. This mutant exhibit alteredpromoter recognition, and thus the mutant is able to initiatetranscription from T7 promoter point mutations that are normallyinactive.

[0006] Ikeda et al. (Ikeda, R. A. et al. Biochemistry, 31:9073-9080,1992 and Ikeda, R. A. et al., Nucl. Acid. Res., 20: 2517-2524, 1992)have described two compatible plasmids that can be used for screeningthe activity of mutated T7 RNAP gene- or promoter sequences. The firstplasmid carries the T7 gene 1 (the gene encoding the T7 RNA polymerase)ligated to an E. coli tac promoter., while the second plasmid carriesthe gene encoding CAT (chloramphenicol acetyl transferase) ligated tothe T7 promoter. E. coli cells carrying these two plasmids are CAM(chloramphenicol) resistant if the T7 polymerase interacts with the T7promoter and transcribes the CAT gene from the second plasmid. If eitherthe T7 promoter or the T7 RNA polymerase is inactive, the CAT gene willnot be transcribed and thus the E. coli cells will be cam sensitive.Ikeda et al. used the plasmids to investigate the effects of certainmutations on the activity of T7 RNA polymerase promoters. With a plasmidsystem like the one described by Ikeda et al., where the T7 RNApolymerase gene 1 is on one plasmid under the control of a suitablepromoter, and the T7 RNA polymerase promoter is on a second plasmidcontrolling a resistance gene like CAT, mutant T7 RNA polymerases itselfcan be screened for their activity as well.

[0007] In vitro transcription with the aid of bacteriophage encoded RNApolymerases (e.g. T7 RNA polymerase, T3 RNA polymerase, and SP6 RNApolymerase) has become a widely applied tool in molecular biology. Nextto the in vitro transcription on its own, as a tool to make fast amountsof RNA bacteriophage, RNA polymerases are part of nucleic acidamplification methods. Such methods are for instance NASBA, 3SR and TMA.In vitro transcription has also been described in combination with PCRas an extra linear amplification step post PCR amplification.

[0008] For all of the above applications it would be advantageous if thereaction temperature could be elevated so that the kinetics of thetranscription reaction becomes better and more importantly thatisothermal amplification methods (NASBA, 3SR and TMA) can be performedat higher temperatures. This higher incubation temperature of theisothermal amplification reaction will enable the amplification ofstructured RNA's more efficiently. Applications where this is importantare amplification of long RNA sequences (>500 nucleotides) and multiplexreactions (i.e. the amplification of multiple RNA sequences in onereaction mixture).

[0009] The present invention relates to mutants of T7 like bacteriophagederived RNA polymerases that have an increased stability.

[0010] Analysis of randomly mutated T7 RNA polymerase mutants revealed anumber of possible mutations that have a stabilizing effect on the T7RNA polymerase protein and enable enzymatic activity at highertemperatures than normal (normal is 37° C.-41° C.). The randomly mutatedT7 RNA polymerase sequences were analyzed by screening the sequences ina two plasmid system as described by Ikeda et al (1992) in Bacillusstearothermophilus. The Bacillus stearothermophilus cells were grown atelevated temperatures (45°-50° C.) and CAM resistance could only beobtained if a mutated T7 sequence would encode a more stable T7 RNApolymerase capable of polymerase activity at these temperatures. In theBacillus stearothermophilus system one plasmid contains an antibioticresistance gene (CAT) under control of the T7 promoter and the otherplasmid contains a mutant library of the T7 RNA polymerase under controlof a Bacillus promoter. In those cases where the mutation allows the T7RNA polymerase to be functional at the elevated temperature the Bacillusstearothermophifus will have become CAM resistant.

[0011] Mutants of the T7 polymerase that have an increased stabilityhave already been described in co-pending, co-owned application numberPCT/EP99/09716, the contents of which are herewith incorporated byreference.

[0012] One of the mutations described in PCT/EP99/09716 was a mutationfrom Serine to Proline at position 633 of the enzyme.

[0013] With the present invention yet further mutations have been foundthat also increase the stability of the enzyme.

[0014] Mutations according to the invention include an F849I, an F880Yand an S430P mutation in the T7 sequence.

[0015] Preferred are those mutants that comprise more than one of thesemutations, preferably in combination with the S633P mutation. Mutantsaccording to the invention may, for example, comprise the S633P mutationin combination with either the F849I mutation or the F880Y mutation.Another mutant according to the invention comprises the F84I mutation incombination with the F880Y mutation.

[0016] Good results with regard to the improved stability were alsoobtained with a mutant comprising the S633P mutation and theF849Imutation as well as the F880Y mutation. The most preferredembodiment of the present invention is a quadruple mutant (mutantcomprising 4 mutations) comprising the S430P, the S633P, the F8491 andthe F880Y mutation.

[0017] This mutant is at least 44 times more thermostable than the wildtype enzyme at 50° C. (having a T½ of 84.5 minutes vs. 1.9 minutes forthe wild type)! Furthermore the specific activity of the quadruplemutant at 50° C. is about 12 times as high as that of the wild typeenzyme at 50° C. (56.8 units/μg for the mutant vs. 4.8 units/μg for thewild type).

[0018] Preferred mutated RNA polymerases according to the invention aremutant RNA polymerases from T7 or SP3 bacteriophages. Due to the highhomology between these enzymes, mutations in the T7 gene 1 sequence arelikely to have the same effect in the corresponding gene sequence of theT3 bacteriophage. Since there is 80% homology between the T7 RNApolymerase and the T3 RNA polymerase the same effects of the 633serine→proline mutation in the T7 gene may be expected for a 634serine→proline amino acid mutation in the T3 RNA polymerase.

[0019] A gene encoding an RNA polymerase, said gene containing one ormore mutations resulting in an increased stability of the encoded RNApolymerase, when compared with the wild type protein is likewise part ofthe present invention, especially where the T7 or T3 RNA polymeraseencoding genes are concerned.

[0020] A serine to proline amino acid change in the protein at position633 of the amino acid sequence of the T7 RNA polymerase is the result ofa T→C mutation at position 1897 of the T7 RNA polymerase nucleotidesequence.

[0021] The mutations are scored compared to the T7 RNA polymerasewild-type sequence as published by Dunn, J. J. and Studier, F. W.[(1983) Complete nucleotide sequence of bacteriophage T7 DNA and thelocations of T7 genetic elements J. Mol. Biol. 166 (4), 477-535] withnumber one being the first nucleotide of the T7 RNA polymerase gene,which is nucleotide number 3171 in the complete genome sequence ofbacteriophage 17.

[0022] The present invention further relates to expression vehicles forthe expression of the mutated RNA polymerases according to theinvention.

[0023] In order to express a gene, the gene is brought under the controlof regulating sequences enabling expression of the protein encoded bysaid gene. Usually, this is done by cloning the gene to be expresseddownstream of such regulating sequences. Regulating sequences enablingexpression of genes or fragments of genes may e.g. be promoter-sequenceseither or not in combination with enhancer sequences.

[0024] These sequences may be the promoter sequences that are found tobe linked to the gene in its native form. Alternatively it may beheterologous promoters. An advantage of using heterologous promoters isthat they offer the possibility to express the gene in host cells thatdo not recognize the gene's native promoter. Moreover, the heterologouspromoter may be a promoter that is inducible, so that expression of thegene can be started at any desired moment.

[0025] Promoter sites are sequences to which RNA polymerase binds,initial to transcription. Promoter-sites exist in a variety of types,i.a. depending on the type of cell, they originate from. Promotersequences have been described for promoters from prokaryotic,eukaryotic, and viral origin. Recombinant DNA molecules of the abovementioned type can e.g. be made by cutting a suitable DNA fragment witha suitable restriction enzyme, cutting a fragment containing regulatingsequences with the same enzyme and ligating both fragments in such away, that the nucleic acid sequence to be expressed is under the controlof the promoter sequence. Many variant approaches to make usefulrecombinants have been described in Sambrook (Sambrook et al, Molecularcloning, a laboratory manual. Cold Spring Laboratory Press, Cold SpringHarbor, N.Y. (1989)).

[0026] In general, recombinant nucleic acid sequences will be clonedinto a so-called vector molecule. The then formed recombinant vectormolecule, often capable of self-replication in a suitable host cell, canbe used to bring the cloned nucleic acid sequences into a cell. This maybe a cell in which replication of the recombinant vector moleculeoccurs. It may also be a cell in which a regulating sequence of thevector is recognised, so that a mutated RNA polymerase according to thepresent invention is expressed. A wide range of vectors is currentlyknown, including vectors for use in bacteria, e.g. pBR322, 325 and 328,various pUC-vectors i.a. pUC 8, 9, 18, 19, specific expression-vectors;pGEM, pGEX, and Bluescript^((R)), vectors based on bacteriophages;lambda-gtwes, Charon 28, M13-derived phages, vectors for expression ineukaryotic cells containing viral sequences on the basis of SV40,papilloma-virus, adenovirus or polyomavirus (Rodriquez, R. L. andDenhardt, D. T., ed.; Vectors: A survey of molecular cloning vectors andtheir uses, Butterworths (1988), Lenstra et al, Arch. Virol.; 110: 1-24(1990)). All recombinant molecules comprising the nucleic acid sequenceunder the control of regulating sequences enabling expression of themutated RNA polymerase are considered to be part of the presentinvention.

[0027] Furthermore the invention comprises a host cell containing anucleic acid sequence encoding the mutated RNA polymerase, or arecombinant nucleic acid molecule encoding the mutated RNA polymeraseunder the control of regulating sequences enabling expression of themutated RNA polymerase.

[0028] The invention also comprises a host cell containing a virusvector containing a nucleic acid molecule encoding the mutated RNApolymerase, or a recombinant nucleic acid molecule encoding the mutatedRNA polymerase under the control of regulating sequences enablingexpression of the mutated RNA polymerase.

[0029] Frequently used expression systems are bacterial, yeast, fungal,insect and mammalian cell expression systems. Such systems arewell-known in the art and easily available, e.g. commercially troughClontech Laboratories, Inc. 4030 Fabian Way, Palo Alto, Calif.94303-4607, USA

[0030] A host cell may be a cell of bacterial origin, e.g. Escherichiacoli, Bacillus subtilis and Lactobacillus species, in combination withbacteria-based vectors as pBR322, or bacterial expression vectors aspGEX, or with bacteriophages. The host cell may also be of eukaryoticorigin, e.g. yeast-cells in combination with yeast-specific vectormolecules, or higher eukaryotic cells like insect cells (Luckow et al;Bio-technology 6: 47-55 (1988)) in combination with vectors orrecombinant baculoviruses, plant cells in combination with e.g.Ti-plasmid based vectors or plant viral vectors (Barton, K. A. et al;Cell 32: 1033 (1983), mammalian cells like Hela cells, Chinese HamsterOvary cells (CHO) or Crandell Feline Kidney-cells, also with appropriatevectors or recombinant viruses.

[0031] Thus, an expression vector comprising a gene encoding an RNApolymerase according to the invention and suitable expression controlsequences is likewise part of the present invention, as well as the hostcells transformed therewith.

[0032] The mutated RNA polymerases according to the invention will findtheir use in all processes where RNA polymerases are normally used andwhere the RNA polymerases, for example, would be used at elevatedtemperatures and thus an improved stability would be advantageous.

[0033] The mutated RNA polymerases according to the invention would beparticularly useful in isothermal transcription based amplificationprocesses for the amplification of nucleic acid. The use of the RNApolymerases in isothermal transcription based amplification methods istherefore also part of the present invention.

[0034] Transcription based amplification techniques involve thetranscription of multiple RNA copies from a template comprising apromoter recognized by an RNA polymerase. With these methods multipleRNA copies are transcribed from a DNA template that comprises afunctional promoter recognized by the RNA polymerase. Said copies areused as a target again from which a new amount of the DNA template isobtained etc. Such methods have been described by Gingeras et al. inWO88/10315 and Burg et al. in WO89/1050. Isothermal transcription basedamplification techniques have been described by Davey et al. in EP323822 (relating to the NASBA method), by Gingeras et al. in EP 373960and by Kacian et al. in EP 408295. Transcription based amplificationreactions may also be performed with thermostable enzymes. Transcriptionbased amplifications are usually carried out at a temperature around 37to 41 Celsius. These thermostable enzymes allow the reaction to becarried out at more elevated temperatures (>41 C.). Such a thermostablemethod is described in EP 682121 filed in the name of Toyo Boseki KK.

[0035] The methods as described in EP 323822, EP 373960 and EP 408295are isothermal continuous methods. With these methods four enzymeactivities are required to achieve amplification: an RNA dependent DNApolymerase activity, an DNA dependent DNA polymerase activity, an RNase(H) activity and an RNA polymerase activity. Some of these activitiescan be combined in one enzyme, so usually only 2 or 3 enzymes arenecessary. Enzymes having RNA dependent DNA polymerase activities areenzymes that synthesize DNA from an RNA template. A DNA dependent DNApolymerase thus synthesizes DNA from a DNA template. In transcriptionbased amplification reactions a reverse transcriptase such as AMV (AvianMyoblastosis Virus) or MMLV (Moloney Murine Leukemia Virus) reversetranscriptase may be used for these activities. Such enzymes have bothRNA- and DNA dependent DNA polymerase activity but also an inherentRNase H activity. In addition an RNase H may be added to the reactionmixture of a transcription based amplification reaction, such as E. coliRNase H.

[0036] The RNA polymerase that is commonly used with transcription basedamplification methods is 7 RNA polymerase. Thus the promoter that isincorporated in the template used for transcribing multiple copies ofRNA would than be the T7-promoter. Usually the template comprising thepromoter has to be created starting from the nucleic acid comprising thetarget sequence. Said nucleic acid may be present in the startingmaterial that is used as input for the amplification reaction. Thenucleic acid present in the starting material will usually contain thetarget sequence as a part of a much longer sequence. Additional nucleicacid sequences may be present on both the 3′- and the 5′-end of thetarget sequence. The amplification reaction can be started by bringingtogether this nucleic acid from the starting material, the appropriateenzymes that together provide the above mentioned activities and atleast one, but usually two, oligonucleotide(s). At least one of theseoligonucleotides should comprise the sequence of the promoter.

[0037] Transcription based amplification methods are particularly usefulif the input material is single stranded RNA, although single or doublestranded DNA can likewise be used as input material. When atranscription based amplification method is practiced on a sample withsingle stranded RNA (of the “plus” sense) with additional sequences onboth the 3′-end and the 5′ end of the target sequence a pair ofoligonucleotides that is conveniently used with the methods as describedin the prior art would consist of:

[0038] A first oligonucleotide (usually referred to a“promoter-oligonucleotide”) that is capable of hybridizing to the 3′-endof the target sequence, which oligonucleotide has the sequence of apromoter (preferably the T7 promoter) attached to its 5′ end (thehybridizing part of this oligonucleotide has the opposite polarity asthe plus RNA used as input material).

[0039] A second oligonucleotide (“primer”) which comprises the 3′ end ofthe target sequence (this oligonucleotide has the same polarity as theplus RNA).

[0040] When such a pair of oligonucleotides, together with all enzymeshaving the appropriate activities, and a sufficient supply of thenecessary ribonucleotides and deoxy-ribonucleotides are put together inone reaction mixture and are kept under the appropriate conditions (thatis, under the appropriate buffer conditions and at the appropriatetemperature) for a sufficient period of time an isothermal continuousamplification reaction will take place.

[0041] The RNA polymerases according to the invention may also be usedin conjunction with other nucleic acid amplification processes. With thePolymerase Chain reaction sometimes primers are used that in which apromoter sequence for a bacteriophage RNA polymerase, especially thepromoter sequence for the T7 RNA polymerase, has been incorporated. Thisenables the transcription of the RNA form the DNA product of the PCRreaction. Again the RNA polymerases according to the invention maylikewise be applied.

[0042] Thus, an enzyme mixture for use in an isothermal transcriptionbased amplification reaction comprising, an RNA polymerase as providedby the present invention, an enzyme having reverse transcriptaseactivity and an enzyme having RNase H activity, is likewise part of thepresent invention.

BRIEF DESCRIPTION OF THE FIGURES

[0043]FIG. 1: Results of transcription assay for the wild type and thequadruple mutant at various temperatures.

The invention is further exemplified by the following examples: EXAMPLE1 Introduction of the Mutation(s) into His-Tagged T7 RNAP Gene on theExpression Plasmid.

[0044] Substitution of mutations was carried out by means ofsite-directed mutagenesis using QuickChange site-directed mutagenesiskit (STRATAGENE). The whole procedure was performed is according to themanufacture's protocol enclosed with the kit. The oligo primers used forintroduction of the serine to proline at amino acid position 633 of T7RNA polymerase mutation are as follows. A: 5′-GTG-TGA-CTA-AGC-GTC-CGG-TCA-TGA-CGC-TGG-3′ B:5′-CCA-GCG-TCA-TGA-CCG-GAC-GCT-TAG-TCA-CAC-3′

[0045] Oligonucleotide B is complementary to oligonucleotide A. Theunderlined sequence indicates the restriction site for Mspl , which isused for screening of mutant clones to contain the oligonucleotidesequences with the T→C mutation at position 1897 of the T7 RNApolymerase nucleotide sequence.

[0046] The primers used to introduce the other mutations are indicatedbelow:

[0047] For S430P (T1288C) substitution: A: 5′-GTT TAC GCT GTC CCA ATGTTC ACC CCG CAA-3′ B: 5′-TTG CGG GTT GTT CAT TGG CAC AGC GTA AAC-3′

[0048] For F849I (T2545A) substitution, (new Sau3AI site appears) A:5′-TTC TAC GAC CAG ATC GCT GAC CAG TTG CAC-3′ B: 5′-GTG CAA CTG GTC AGCGAT CTG GTC GTA GAA-3′

[0049] For F880Y (T2639A) substitution, (New Miul site appears) A:5′-TCT TAG AGT CGG ACT ACG CGT TCG CGT AAC-3′ B: 5′-GTT ACG CGA ACG CGTAGT CCG ACT CTA AGA-3′

[0050] PCR reaction mixture and conditions were as follows. 10x Pfubuffer 5 μl Oligonucleotide A (100 ng/μl) 1.25 μl Oligo B 1.25 μl 2 mMdNTPs 1.25 μl plasmid template* 1 μl H2O 41 μl total 50 μl

[0051] The plasmid template contains the complete T7 RNA polymerase wildtype gene sequence as published in the databases (Dunn, J. J. andStudier, F. W. (1983) Complete nucleotide sequence of bacteriophage T7DNA and the locations of T7 genetic elements J. Mol. Biol. 166 (4),477-535) fused to a histidine tag for simple purification in laterprocedures. T7 RNA polymerase gene was cloned by PCR using T7 DNA (SigmaD4931) as a template. The PCR-amplified T7 RNA polymerase DNA was thencloned into appropriate restriction site of pUC18(tag) plasmid which wasmade in advance by inserting tag sequence into the multiple cloning site(MCS) of pUC18. After making sure the DNA sequence of T7 RNAP gene wasinserted by sequencing, the Tag-T7 RNA polymerase fusion gene wassubcloned into appropriate site of pKK223-3 expression plasmid(Pharmacia Biotech 27-4935-01) to make Tag-T7RNAP/pKK223-3.

[0052] The PCR reaction was performed with the following temperaturecycling protocol:

[0053] 95° C. 30sec

[0054] 55° C. 1 min

[0055] 68° C 14min/18cycles

[0056] After the PCR reaction, 10units of Dpnl restriction enzyme wasadded and incubated at 37° C. for 1 hr. One μl of Dpnl-treated DNA wasthen used for transformation of E. coli JM109. Finally, the mutant T7RNA polymerase clone was isolated by screening the plasmid DNA using theMspl restriction enzyme.

EXAMPLE 2

[0057] Mutant T7 RNA polymerase gene(s) containing plural mutations(amino acid substitutions) was constructed by substituting therestriction fragment containing a mutation for the same fragment of themutant gene which contains other mutation(s). The restriction sites usedfor substitution were as follows:

EXAMPLE 3 In Vitro Transcription Assay

[0058] Reaction mixture for the in vitro transcription assay is asfollows: 10 × T7 RNAP buffer 5 μl rNTP(each 25 mM) 0.8 μl 0.1% BSA 5 μlRnase inhibitor (40 units/μl) 0.5 μl Template plasmid (0.5 μg/μl) 1 μlT7RNAP 25 units H2O/total 50 μl

[0059] Incubate at each temperature for 60 minutes

[0060] Apply 3 μl of above reaction mixture on 0.7% agarose gel.

[0061] Results are depicted in FIG. 1.

EXAMPLE 4 Comparison of Specific Activity of W.T. and Quadruple mutant(F880Y+F8491I+S633P+S430P)

[0062] The following protocol is used to determine the enzymatictranscription activity of T7 RNA polymerase. (For (For 10 1 assay)assays) 10xtranscription buffer (*2) 5 μl 50 μl 100mM rNTP mix (25mMeach rNTP) 0.8 μl 8 μl T7 DNA (Sigma D4931)(0.5 μg/μl) 2 μl 20 μl BSA(1mg/ml) 2.5 μl 25 μl H20 34.2 μl 342 μl [3H] rUTP (NEN: NET-287) 0.5 μl 5μl total 45 μl 450 μl

[0063] In this assay, transcription activity is calculated by using thefollowing formula:

Activity (units/μl)=[cpm(Sample)−cpm(Blank)]×24/cpm(Total)

[0064] (1 unit is defined as a activity to catalyzes the incorporationof 1 nmole of labelled nucleotidetriphosphate into acid-insolublematerial in 60 minutes) (*1) if necessary, dilute the enzyme solution to0.5-4 units/μl with dilution buffer Dilution buffer: 20 mM KPO₄ (pH 7.5)100 mM NaCl 0.1 mM EDTA 1 mM DTT 50% (VN) Glycerol (*2) 10 ×transcription buffer 400 mM Tris-HCl (pH 8.0) 200 mM MgCl₂ 50 mM DTT

[0065] Results are depicted in Table 2: TABLE 2 Specific activity(units/μg protein) Reaction Temperature W.T. Q-mutant 37 22.0 29.9 4026.9 35.9 45 32.2 49.6 50 4.8 56.8 55 1.6 8.8 60 0.0 3.3 65 0.0 0.9

EXAMPLE 5

[0066] In this example the half life T_(1/2), of different T7 RNApolymerases is determined using the following protocol:

[0067] 1. Prepare the following reaction mixture (For I assay) 10xtranscription buffer 10 μl 0.5 M KCl 14 μl BSA (1 mg/ml) 10 μl H2O 56 μltotal 90 μl

[0068] TABLE 1 T_(½) compared between T7 wild-type and T7 mutants T ½(min) SA (units/μg At 50.0 At 48.0 At 46.0 Mutation protein Test 1 Test1 Test 2 Test 1 WT 24.9 1.9 2.1 16.6 S430P 6.8 S633P 38.7 9.3 9.0 58.3F8491I 39.2 9.8 53.3 F880Y 32.4 8.4 42.7 S633P + F849I 31.7 6.7 46.0S633P + F880Y 35.3 4.5 49.3 F8491I + F880Y 33.8 5.9 37.8 S633P + F849I +35.0 28.0 58.0 F880Y 84.5

EXAMPLE 6 Establishment of Large Scale Production System of T7 RNAPEnzyme.

[0069] To obtain large quantity of enzyme, conditions for cultivating E.coli and purification of T7 RNAP enzyme were examined. Conditionsfinally determined were as follows. For purification conditions,reference is made to a publication by Studier (P.N.A.S., USA, 81,2035-2039, 1984.

[0070] 6.1: Procedure for cultivating E. coli harboring T7 RNAPexpression plasmid.

[0071] 1. Inoculate single colony E. coli BL21 harboring T7 RNAPexpression plasmid into 2 ml of LB medium and cultivate at 30° C. for 16hours (seed seed culture).

[0072] 2. Inoculate 1 ml of seed culture into 100 ml of LB medium andcultivate at 30° C. for 16 hours (seed culture).

[0073] 3. Inoculate 100 ml of seed culture into 6 L of TB medium andcultivate at 37° C. for 10-12hr (main culture). (All medium usedcontains 50 μg of ampicillin per ml.) LB medium: Tripton peptone (Difco)10 g Yeast Extract (Difco) 5 g NaCl 10 g/L Adjust pH 7.5 with NaOH TBmedium: Tripton peptone (Difco) 12 g Yeast Extract(Difco) 24 g KH₂PO₄2.31 g K₂HPO₄ 12.5 g Glycerol 4 ml/L

[0074] The results are given in Table 2: TABLE 2 Cultivation time(hours) OD 660 PH 0 0.75 6.96 2 2.45 6.81 4 5.75 6.7 6 7.35 6.44 8 12.36.83 10 16.6 7.47 12 19.15 8.05 14 19.5 8.35 16 18.75 8.49 18 17.2 8.5420 16.3 8.57 22 15.05 8.56

[0075] 6.2 Procedure for purification of T7 RNAP.

[0076] 1. 100 g of E. coli cells was suspended in 500 ml of buffer A anddisrupted by Frenchpress. This lysate was then centrifuged andsupernatant (crude extract) was obtained.

[0077] 2. To the crude extract, 16.9 ml of 5% Polyethylenimine (PEI)solution was added to precipitate nucleic acids. Supernatant wasobtained by centrifugation.

[0078] 3. To the supernatant after PEI treatment, ammonium sulfate wasadded to final 45% saturation. The mixture was stirred at 45° C. for 30min to allow precipitation. The ammonium sulfate precipitate wascollected by centrifugation, dissolved in 200 ml of buffer B (+50mMNaCl), and then dialyzed against the same buffer.

[0079] 4. The enzyme solution was loaded on 100 ml column of Affi-Gelblue (Bio-Rad). The column was washed with buffer B (+100 mM NaCl), andthe protein was eluted with buffer B (+2M NaCl). The peak fractions werepooled.

[0080] 5. To the collected pool, ammoniumsulfate was added to final 45%saturation. The ammonium sulfate precipitate was then collected,dissolved in 200 ml of Buffer B (+25 mM NaCl), dialyzed against the samebuffer, and loaded on 200 ml column of DEAE-cellulofine column. Thecolumn was washed with buffer B (+25 mM NaCl), and the protein waseluted with 1000 ml of gradient form 25 to 150 mM NaCl. The peakfractions were collected.

[0081] 6. To the collected pool, ammoniumsulfate was added to final 45%saturation. The ammonium sulfate precipitate was then collected,dissolved in 30 ml storage buffer and dialyzed against the same buffer.The resulting enzyme was finally filtrated and stored at −300° C. BufferA: Buffer B: Storage buffer 50 mM Tris-HCl (pH 8.0) 20 mM KPO₄ (PH 7.7)20 mM KPO₄ (pH 7.7) 1 M NaCl NaCl 100 mM NaCl 2 mM EDTA 1 mM EDTA 0.1 mMEDTA 1 mM DTT 1 mM DTT 1 mM DTT 5% Glycerol 50% Glycerol 0.01%TritonX-100

[0082] Results are depicted in Table 3: TABLE 3 Summary of purification:Total Protein Total Speicifc Vol. Activity Activity Conc. proteinactivity Step (ml) (Units/μl) (KU) (mg/ml) (mg) (units/μg) 1 CrudeExtract 845 ND — 24.6 20787 — 2 After PEI 869 ND — 23.4 20124 — 3 Aftersalting out 200 805.1 161012 36.5 7300 22.0 4 After Affi-gel blue 524312.3 163619 5.2 2724 60.1 5 After DEAE cellulo. 220 354.3 77950 3.8 83693.2 6 After salting out 37 1865 69025 19.2 710 97.2 and dialysis

[0083] 6.3 Quality Control Assays for Purified 7 RNAP

[0084] The purified enzyme was tested by the following Quality ControlAssays and the in vitro transcription assay.

[0085] Functional Absence of Exonuclease Activity:

[0086] 120 units of the purified enzyme was incubated with 10 μl of3H-E. coli (NET-561) at 37° C. for 4 hours. The release of acid solubleDNA is less than 0.01% /units/hour

[0087] Functional Absence of Endonuclease Activity:

[0088] 60 units of the purified enzyme is incubated with 1 μg of phiX174DNA at 37° C. for 4 hours. No visible change is detected in band patternupon agarose gel electrophoresis.

[0089] Functional Absence of Rnase Activities:

[0090] 200 units of the purified enzyme is incubated with 2 μg of16S&24S ribosomal RNA (Boehringer) at 37° C. for 4 hours. No visiblechange is detected in band pattern upon agarose gel electrophoresis.

[0091] Performance of Transcription Reaction.

[0092] Each 30 units of the purified enzyme and a commercially availableRNAP were used in the following in the vitro transcription assaydescribed above.

[0093] Mixtures were incubated at both 37 and 48 degrees Celsius for 1hour. The quantity of RNA produced by the purified enzyme at 48 degreesCelsius was almost the same as the quantity of RNA produced by thecommercially available enzyme ate 37 degrees.

1 8 1 30 DNA Artificial sequence Synthetic oligonucleotide 1 gtgtgactaagcgtccggtc atgacgctgg 30 2 30 DNA Artificial sequence Syntheticoligonucleotide 2 ccagcgtcat gaccggacgc ttagtcacac 30 3 30 DNAArtificial sequence Synthetic oligonucleotide 3 gtttacgctg tcccaatgttcaacccgcaa 30 4 30 DNA Artificial sequence Synthetic oligonulceotide 4ttgcgggttg aacattggca cagcgtaaac 30 5 30 DNA Artificial sequenceSynthetic oligonulceotide 5 ttctacgacc agatcgctga ccagttgcac 30 6 30 DNAArtificial sequence Synthetic oligonucleotide 6 gtgcaactgg tcagcgatctggtcgtagaa 30 7 30 DNA Artificial sequence Synthetic oligonucleotide 7tcttagagtc ggactacgcg ttcgcgtaac 30 8 30 DNA Artificial sequenceSynthetic oligonucleotide 8 gttacgcgaa cgcgtagtcc gactctaaga 30

1. A T7 RNA polymerase that is mutated when compared with the wild typesequence, said mutant having an improved stability, said mutantcomprising at least one of the mutations selected from the groupconsisting of S430P, F8491and F880Y.
 2. An T7 RNA polymerase accordingto claim 1, said T7 RNA polymerase also being mutated at least in thatit has a serine to proline amino acid change on position 633 of theamino acid sequence of said T7 RNA polymerase.
 3. An T7 RNA polymeraseaccording to claim 1, characterized in that said mutant comprises two ormore mutations selected from said group.
 4. An T7 RNA polymeraseaccording to claim 2 comprising at least the S633P, the F849I and theF880Y mutations.
 5. A T7 RNA polymerase according to claim 4, comprisingthe S430P, the S633P the F849I and the F880Y mutations.
 6. A geneencoding an RNA polymerase, said gene encoding a mutated T7 RNApolymerase according to claim
 1. 7. An expression vector comprising agene according to claim 6 and suitable expression control sequences. 8.A cell transformed with a vector according to claim 7, and capable ofexpressing the mutated RNA polymerase.
 9. Use of an RNA polymeraseaccording to any of claims 1-5 in an isothermal transcription basednucleic acid amplification reaction.
 10. An enzyme mixture for use in anisothermal transcription based amplification reaction comprising, An RNApolymerase according to any of claims 1-5, An enzyme having reversetranscriptase activity and optional Rnase H activity